Glucose oxidase (β-D-glucose: oxygen 1-oxidoreductase; EC1.1.3.4) specifically catalyzes oxidation of β-D-glucose to gluconic acid and simultaneously production of H2O2 by using O2 as an electron acceptor. Glucose oxidase was firstly discovered in the extracts from Aspergillus niger by Muller's lab in 1928. Glucose oxidase is wildly produced by animals, plants and microorganisms, and most researches focus on microbial glucose oxidase. Microbial glucose oxidase mainly exists in A. niger and Penicillium spp. Industrial glucose oxidase is also obtained by these two natural producing strains. However, the production and enzymatic activity of glucose oxidase originally expressed from the natural sources are not good enough. Besides, A. niger and Penicillium spp. simultaneously produce glucose oxidase and other kinds of proteins that cause the difficulty in protein purification during industrial producing process, and finally lead to high cost of industrial production. Hence, there are more and more researches about the production of glucose oxidase by other microbial expression systems, especially by Pichia pastoris which is commonly used in industrial applications.
Glucose oxidase is widely used in many industrial applications for years. It can be used as an antioxidant to preserve food and maintain the flavor of beer in food industry. Besides, it is one of bread/dough-improving additives in baking industry. Glucose oxidase is also used as a glucose biosensor which measures blood glucose levels for diabetes monitoring, for example. Additionally, H2O2 produced by glucose oxidase is capable of bleaching in textile industry. In recent years, glucose oxidase is extensively used in feed industry. It can reduce oxygen levels, produce H2O2, and decrease environmental pH value because of produced gluconic acid, and thus can further inhibit the growth of some bacteria or fungi. Therefore, glucose oxidase is used as a feed additive to improve the gut environment in animals. Moreover, glucose oxidase is possible to be used in biofuel production. To sum up, glucose oxidase plays a critical role in many different industrial applications. Thus, there are increasing researches about enhancement of its catalytic efficiency and protein production and improvement of its protein properties such as thermostability and pH stability.
Currently, many researches try to obtain better enzymes by either screening in nature or modifying present enzymes. In the present invention, glucose oxidase is modified by rational design to increase its thermostability, so as to further increase its application potential and economic value in industry.